The arrangement of rhodopsin's polypeptide chain in the rod outer segment disk membrane will be studied by proteolysis on the outside and inside membrane surface. The proteolytic fragments will be subject to further chemical cleavage by amino acid specific cleaving agents. The amino acid composition of the fragments will be determined as well as their position in the linear sequence. The polypeptide conformation of the fragments will be studied after reincorporation into lipid bilayers using circular dichroism. The complete amino acid sequence of bovine rhodopsin will be determined via sequencing of the complementary DNA (c DNA) to the rhodopsin messenger RNA by cloning the rhodopsin sequence to an E. coli plasmid. Lipid-rhodopsin interactions will be studied by deuterium nuclear magnetic resonance (NMR) using specifically deuterated phospholipids containing polyunsaturated or mono-unsaturated fatty acid chains. Proton and carbon-13 NMR, rhodopsin bleaching kinetics, as well as neutron and x-ray diffraction will be studied as a function of lipid/protein ratio and polyunsaturation vs. mono-unsaturation in recombinant membranes and the results compared with the intact native membrane. A goal is to understand the role of the polyunsaturated fatty acids in the ROS.